This invention relates to methods and apparatus for manufacturing ethanol and other alcohols from sugar solutions which may be obtained from the hydrolysis and breakdown of many agricultural and biological residues and wastes. More particularly, this invention relates to the manufacturer of such alcohols by the use of immobilized cell reactors.
In this period of critical energy shortages, the nations attention is being directed toward the development of alternate or plentiful sources of energy. Particularly, as world reserves of petroleum are depleted, new sources of carbon and hydrogen must be found to supply mankinds chemical and energy needs. While our present sources of petroleum are being rapidly depleted, it will be appreciated that in most parts of the world large quantities of biological residue materials are available which can be converted to suitable chemicals. For example, it has been estimated that the United States alone has about three hundred million dry tons a year of agricultural residues. In particular, corn stover accounts for about one-half of the total U.S. agricultural residue. At present, it has been estimated that this corn residue alone could supply all of the petro chemical needs of the United States if a conversion efficiency as low as forty percent can be obtained. But, in addition to the corn stover there are, of course, many other biological and agricultural residues including corn cobs, pine and oak bark, wheat, straw, and any and all other types of animal vegetable and plant matter. Thus, even though the percentage of conversion will vary, the primary constituents of all such plant material are well recognized as being starch, cellulose, hemicellulose and lignin. The hemicellulose and cellulose fractions can be converted into energy or chemicals by direct combustion (i.e. burning), pyrolysis or biological conversion. To date, direct combustion has been the most commonly used. But, biological conversion is much preferable because of the higher efficiency and the preservation of minerals and nutrients which can be returned to the soil. However, to be able to convert these agricultural residues by biological means it will be necessary to hydrolize the starch, cellulose or hemicellulose into monomeric sugars such as xylose, pentoses, hexoses, and galactoses. These simple sugars can then easily be converted into alcohols, acids, aldehydes or gases depending upon the type of microorganism selected for the biological conversion.
The most common way of converting various sugars such as xylose and glucose into ethanol has been by batch fermentation and continuous stirred tank fermentation (CSTF). Unfortunately, both of these techniques are slow and are quite susceptable to inhibition of growth from materials in the substrate or in the product. Typically, for example, the batch fermentation vats are drained and cleaned every two or three days and hence are out of production 25% to 30% of the time. Because of these difficulties, there is a definite need for an efficient technique of converting simple sugars such as glucose and xylose into ethanol. The simple sugars are easily obtained by hydrolyzing cellulose and hemicellulose containing materials such as corn stover and the like. For example, Dr. James Gaddy at the University of Missouri in Rolla discovered that the reaction rate of glucose fermentation to ethanol could be increased nine (9) times if the yeast was attached to ceramic Raschig Rings rather than put into the solution and run continuously. In addition, there was no washout, as the yeast stayed in the reactor. Gaddy proposed columns 12 ft. in diameter and 50 ft. high filled with Raschig Rings. No mixing is possible after inoculation. The applicant of the present invention has an application now pending in the Patent Office which describes a high area ceramic contactor which should provide even better rates than the Raschig Rings. In an experiment by the applicant in which string was used to suspend a ceramic high area contactor in a nutrient solution, it was discovered that significantly more bacterial growth adhered to the string than to the ceramic. Thus, it is seen that techniques for immobilizing various enzymes by attachment to water insoluble materials has received considerable attention recently. However, as is pointed out in volume 261, of the Nature Magazine, dated May 20, 1976, a logical extension of this approach of immobilizing enzymes is the immobilization of other microorganisms which are often themselves the source of enzymes. The advantage of immobilizing the microorganism itself is obvious since the time consuming procedures for enzyme extraction and purification are eliminated, and cofactors and coenzymes are readily available. Further, the cellular enzymes are often organized into the requisite metabolic pathways such that problems associated with the enzyme instability may be avoided. In addition, the use of immobilized cells avoids the problems in industrial processes of separating the product from the enzyme. Early techniques of immobilization of cells included the standard glutaraldehyde procedure and entrapment in a polyacrylamide gel. Unfortunately, use of polyacrylamide gel produces a minimum interaction between the microbial cell and the nutrients in the medium. This maximizes the probability of cell survival, but the availability of the cell to any intended nutrients is reduced can only reach the cell by diffusion. As noted in Advances in Biochemical Engineering, volume 5. Jack and Zajic found that "cells lying deeper than 0.35 mm inside the polyacrylamid gel were being limited by oxygen deprivation." According to the "Nature" article mentioned above, an alternate approach of immobilizing cells on collagen is by the formation of a stable network of multiple ionic linkages, hydrogen bonds and Van der Walls interreactions which successfully immobilizes the cells on the hydroxides of titanium and zirconium by a chelation process. In addition there have been numerous other techniques discovered for immobilizing various bacteria.
As an example, refer to U.S. Pat. No. 4,138,292 issued to Chibata on Feb. 6, 1979 which teaches an enzyme or microorganism entrapped within the gel matrix of a sulfated polysaccharide in the presence of an ammonium ion, a metal ion, a water soluble amine or a water miscible organic solvent. Also, referring to U.S. Pat. No. 4,206,259 by Rohrback et al, there are disclosed support matrices for immobilized enzymes. More specifically, the patent shows support matrices consisting of an organic-inorganic composite in which the inorganic support material is combined with an organic copolymer prepared in situ and entrapped within the pores of the organic support. The copolymer is formed by the reactions of aminopolystyrene with a sufficient excess of bifunctional monomer to provide a copolymeric product containing terminally functionalized groups capable of covalently binding the enzymes at the terminal reactive portions. In a particular example, alumina base having a particle size from 25 to 40 mesh is added to 10 millimeters of five percent weight by volume of aminopolystyrene and 0.1 M hydrochloric acid. After standing one hour, the mixture is degased and filtered and the support containing the aminopolystyrene is dried. This composite is mixed with a 1.5 percent aqueous solution of glutaraldehyde. After a setting period the remaining organic-inorganic material is washed with water. The final immobilized conjugate is then treated with a commercial glucoamylase for 16 hours while maintaining the temperature of the composite at 4.degree. C.
From the above, it is seen that the present methods of immobilizing bacteria are quite complex. An effective or simple attachable method would be a great help. Therefore, it is an object of this invention to provide apparatus for readily attaching bacteria. Although various types of bacteria may be used to hydrolize cellulose to sugar which is then converted to alcohol, there are, of course, various types of such operatives which are superior to others. For example, the bacterium Clostridium thermocellum can hydrolize cellulose to sugar and sugar to alcohol in one step, and research at Massachusetts Institute of Technology has resulted in a method of producing butanol by use of Clostridum Acetobutylium. Furthermore, Dr. Wang at MIT has reported that special strains of two bacteria produced by genetic mutuation produce ethanol from cellulose and are tolerant to alcohol. Unfortunately, these particular bacteria can be used only with an inorganic support such as ceramic. Such bacteria would attack a support of wood or certain other organic materials.
U.S. Pat. Nos. 3,983,000, 4,071,409, 4,149,936, 4,149,937, 4,153,510 by Messing describe methods of attaching microbes to glass. Other, particularly suitable organisms include certain aerobic species of Pseudomonas from the family Pseudomonadaceae, certain endospore forming species of Clostridium from the family Bacillaceae, certain species of Actinomycetaes and Streptomyces from the family Streptomycetaceae. Finally perhaps the most promising is the anaerobic rod Zymomonas mobilis (Z. mobilis) from the family Vibrionaceae.
As has been mentioned, ethanol is a very useful alternate liquid transportion fuel which can either be blended with gasoline (gasohol) to extend present supplies or used directly by modifying the internal combustion engine. As mentioned heretofore, Z. mobilis has been found to be particularly effective in the production of ethanol. In comparing the kinetics of alcohol production with an anaerobic bacterium such as Zymomonas mobilis and a yeast Saccharomyces cerevisiae, the Z. mobilis shows favorable results. Likewise, studies and comparisons of Z. mobilis with yeast indicate that the glucose uptake rate is two or three times faster than yeast, and that Z. mobilis gave yields of up to 97% of the theoretical value. Further, genetic manipulation is simpler with bacteria such as Zymomonas species than with yeasts. This opens up the possibility of extending the range of nutrient utilization. The use of Z. mobilis with a specially designed fermentator should have considerable promise in minimizing the pollution problem associated with fermentation. New yeasts are being developed, however, Kluyreromyces fragilis ferments lactose.
The use of reaction towers and packed column reactors and other means for supporting a biomass reaction, has also received considerable attention; however, problems immediately surface whether Rashing Rings or Clyde High Area Contactors are used. For example, U.S. Pat. No. 4,127,447 issued to Griffith et. al., on Nov. 28, 1978 is concerned with the problems of a column being plugged by the build up of anaerobic microorganisms attached to the supports in an upflow packed bed. To limit this growth rate, a membrane disrupting detergent is provided in the column to lyse the dead microorganisms for making them available as nutrients for the live organisms. In addition, the growth may be restricted by limiting the availability of essential nutrients and/or by providing the presence of predatory protozoa which consume the anaerobic microorganisms.
Still another U.S. Pat. No. 3,878,301 issued to Berdelle-Hilge on Apr. 15, 1975 discloses a process of controlling the productivity of microoganisms which are held in at least one reaction bed through which nutrients flow with a short contact time. The process is characterized in that the desired productivity is obtained by deliberate quantitative and/or qualitative modification of the contact conditions between the nutrients and the microorganisms.
In a similar manner, Mr. Coulthard in his U.S. Pat. No. 3,981,803 issued Sept. 21, 1976 in which he describes an apparatus for achieving anaerobic fermentation notes the problem of the large biomass build up, but provides no solution.
However, it is suggested that restricting the growth of the biomass, or controlling the contact conditions or the nutrients or providing predatory protozoa is not the answer. Therefore, a technique for allowing uninhibited growth of the biomass for achieving the rapid conversion of sugars to alcohol is desirable; while at the same time the techniques for controlling and preventing the plugging of the reactor by the excess amounts of biomass is necessary. In recent tests by the Applicant for determining the build up of Z. mobilis on a Clyde High Area Contactor suspended in a solution by a string, as was mentioned earlier, it was noticed that a greater build up of bacteria occurred on the support string than on the contactor. Further experimentations showed that the build up of Z. mobilis was particularly great on such substrates as string, wood and netting.
Therefore, it is an object of the present invention to disclose a fermenter which encourages the growth of a selected microorganism, but in which plugging or stoppage of the fermenter by excess biomass is eliminated.
It is another object of this invention to describe a fermenter in which excess growth is controlled by mechanical or physical discharge of the biomass.
It is yet another object of this invention to describe a fermenter, wherein the excess biomass is controlled by means other than limiting the nutrients of the biomass or using predatory protozoa.
It is yet another object of this invention to describe a fermenter wherein CO.sub.2 can leave without disturbing the rest of the system during the production of alcohol.
It is yet another object of this invention to describe a fermenter in which bacteria or other microorganisms easily attach to a substrate having a large area.
It is still another object of this invention to disclose a fermenter suitable for the production of alcohol and other chemicals.
It is yet another object of this invention to describe a fermenter useful in the treatment of waste water.
It is another object of this invention to provide a method for removing excess biomass from heating tubes so as to maintain efficient heat transfer. To accomplish the above mentioned objects as well as other objects which will become evident from the following detailed description, the present invention discloses apparatus which according to one embodiment comprises a container suitable for holding a selected nutrient. The container includes a port for receiving the nutrient medium, exhaust ports for discharging excess organisms and CO.sub.2, and a port for removing the resulting chemical such as ethanol. A substrate comprised of a multiplicity of fibers such as string or ceramic is provided for supporting the growth of a selected microorganism such as for example Z. mobilis. The immobilizing substrate is itself supported in a desired position within the container by a support means to achieve proper flow of materials. There is also included means for physically detaching the organism from the immobilizing substrate such as by striking or ultrasonic waves. The use of Z. mobilis as the organism and glucose as the fermentable carbon source in the medium is especially useful for the production of ethanol.